Ras homolog gene family member A(RhoA)is a small GTPase protein.Rho-associated coiled-coil forming protein kinase(ROCK)can be activated during the occurrence and development of ischemic stroke.RhoA/ROCK signaling pathway is an important regulatory factor in the pathological process of ischemic stroke,and regulation of this signaling pathway has become a research focus in promoting neuronal recovery and improving cerebral ischemia/reperfusion injury after ischemic stroke.However,only Fasudil is currently available as a RhoA/ROCK inhibitor.The rest is still in the stage of research and development or clinical trials,and has broad research prospects.In this paper,the regulatory role and mechanism of RhoA/ROCK signaling pathway in ischemic stroke were analyzed,and the application of inhibitors and regulatory drugs were described,aiming to provide new ideas for the prevention and treatment of ischemic stroke.

Osteoporosis （OP） is a systemic metabolic bone disease. Amid population aging， OP has become a major health problem for the middle-aged and the elderly in China. Aging， iron load， and estrogen deficiency break the balance between oxidation and antioxidant systems， and the increase of reactive oxygen species mediates oxidative stress to damage DNA， lipids， proteins and other macromolecules， thus accelerating cell apoptosis and inducing OP， obesity， and neurodegenerative disorders. It has been found that oxidative stress is of great significance in the pathogenesis of OP. Oxidative stress regulates the signaling pathways， cytokines， and proteins related to the mesenchymal stem cells， osteoblasts， and osteoclasts， thereby weakening the osteogenic differentiation of mesenchymal stem cells， inhibiting osteoblast mineralization， and promoting the activation， proliferation， and maturation of osteoclasts. As a result， the dynamic imbalance between bone resorption and bone formation occurs， influencing bone remodeling and promoting the progression of OP. At the moment， anti-bone resorption drugs， bone formation-promoting drugs， and hormones are mainly used in clinical settings in western medicine. However， due to the long treatment cycle and the occurrence of serious gastrointestinal reactions， hypocalcemia， osteonecrosis， and others， patients show poor compliance and thus the effect is not as expected. Traditional Chinese medicine （TCM） demonstrates remarkable effect on OP attributing to the multi-pathway and multi-target characteristics. With low price and few adverse reactions， TCM is widely applied in clinical practice in comparison with western medicine. TCM has unique advantages in the treatment of OP by regulating oxidative stress. It exerts the therapeutic effect on OP by modulating different signaling pathways， providing new mindset for the treatment of this disease. Therefore， through literature research， this study summarized the research on mechanism of oxidative stress in OP and the treatment by TCM， which is expected to lay a foundation for further research.

AIM: To study the effects of trigliptin succinate on gut microbiota of type 2 diabetic mice. METHODS: 16S rRNA high-throughput sequencing method was used to sequence the intestinal flora of mice in the healthy group, the T2DM group, the trigliptin succinate group and the sitagliptin phosphate group. QIME was used to filter the data, classify and annotate the species. Alpha diversity index and Beta diversity index of the samples were analyzed.The richness and diversity of bacteria in the four groups were compared. RESULTS: The gut microbiota structure of mice in the healthy group, the T2DM group, the trigliptin succinate group and the setagliptin phosphate group were significantly different. The results showed that the ratio of Firmicutes to Bacteroidetes was decreased compared with that in the healthy group. Cyanobacteria, Verrucomicrobia and Tenericutes had significant differences (P< 0.05). Potential biomarkers for T2DM group were Bacilli, Lactobacillales, Lactococcus and Streptococcaceae. Candidate biomarkers of trigliptin succinate group may be Bacteroidia, Bacteroidetes, Bacteroidales, Prevotella, Paraprevotellaceae, Parabacteroides, Porphyromonadaceae; The candidate biomarkers of sitagliptin phosphate group may be Lactobacillus, Lactobacillaceae and Helicobacter. CONCLUSION: The intestinal flora of mice in the trigliptin succinate group was significantly different from that in the healthy group and the T2DM group. Using trigliptin succinate to improve the intestinal flora of mice might achieve the hypoglycemic effect by improving the intestinal flora.

Objective:To screen out the potential key genes of sepsis-associated acute kidney injury (AKI), and provide theoretical and experimental evidence for the treatment of sepsis-associated AKI.Methods:① Bioinformatics analysis: two gene expression datasets (GSE30718 and GSE53773) were downloaded for bioinformatics analysis from the Gene Expression Omnibus (GEO). These two datasets recorded mRNA microarray data from kidney biopsies before and after kidney transplantation, and a subset of patients developed AKI after kidney transplantation. Differential analysis was conducted, and the genes with the same differential expression and a higher area under the receiver operator characteristic curve (AUC) in both databases were used as the target gene for subsequent cell experiments. ② Cell validation experiment: human proximal renal tubular cells HK2 were cultured in vitro, and lipopolysaccharide (LPS) was used for establishing LPS-HK2 cell model (LPS 10 mg/L for 6 hours, LPS model group), and the blank control group was set. Then, small interfering RNA (siRNA) technology was used to knock down the target gene obtained by bioinformatics analysis in LPS-HK2 cells (gene knockdown group), and a gene negative control group was set. The real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR) technique was used to detect the expression of the target gene in HK2 cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors in the cell supernatants. Western blotting was used to detect the expressions of key apoptosis proteins. Results:① Results of bioinformatics analysis: 325 genes in the two datasets showed the same expression trend, of which 144 were significantly down-regulated and 181 were significantly up-regulated, while the expression difference of secretory leukocyte protease inhibitor (SLPI) in the two datasets was both statistically significant. Further receiver operator characteristic curve (ROC curve) analysis confirmed that the SLPI expression in GSE30718 and GSE53773 datasets had a high diagnostic efficiency for AKI, with AUC of 0.83 and 0.92, respectively. Therefore, SLPI was selected as the target gene for subsequent cell validation experiment. ② Cell validation experiment: the RT-qPCR analysis showed that the expression of SLPI in LPS-HK2 cells of the LPS model group was significantly higher than that of the blank control group (2 -ΔΔCT: 1.80±0.14 vs. 1.00±0.11, P < 0.01), and the change trend was the same with the results of bioinformatics analysis. Furthermore, knockdown SLPI gene analysis showed that the levels of inflammatory factors in LPS-HK2 cells supernatants in the gene knockdown group were significantly higher than those in the negative control group [Interleukin-6 (IL-6, ng/L): 509.58±27.08 vs. 253.87±75.83, IL-1β (ng/L): 490.99±49.52 vs. 239.67±26.97, tumor necrosis factor-α (TNF-α, ng/L): 755.22±48.66 vs. 502.06±10.92, all P < 0.01]. The above results indicated that SLPI could inhibit the inflammatory response of HK2 cells induced by LPS. The expressions of key apoptosis proteins Bax and caspase-3 in LPS-HK2 cells in the gene knockdown group were significantly higher than those in the negative control group [Bax protein (Bax/GAPDH): 1.38±0.12 vs. 1.00±0.10, caspase-3 protein (caspase-3/GAPDH): 1.44±0.15 vs. 1.00±0.11, both P < 0.05], and Bcl-2 expression was significantly decreased (Bcl-2/GAPDH: 0.83±0.08 vs. 1.00±0.05, P < 0.05), the above results indicated that SLPI could inhibit the apoptosis of cells in the inflammatory response. Conclusion:SLPI can inhibit the inflammatory response and apoptosis of HK2 cells induced by LPS, which may be involved in the protective mechanism of renal tubular cells in the response to sepsis, and is a potential target for the treatment of sepsis-associated AKI.

【Objective】 To identify and propose blood transfusion suggestions for 3 children suspected to have red blood cell T polyagglutination. 【Methods】 According to the RBC reactions with phytohemagglutinin, adult serum and cord blood serum, aggregation test with polybrene reagent and MN antigen phenotype test were carried out on 3 children to confirm the presence of T polyagglutination. The donor serum with negative or weak reactions was selected by minor cross matching for the 3 children who needed therapeutic plasma exchange(TPE). 【Results】 Three cases of RBC T polyagglutination were caused by bacterial infection, with transient appearance of MN antigen; the samples were reactive to peanut agglutinin, soybean agglutinin, adult serum but nonreactive to cord blood serum, and didn′t aggregate after adding polybrene reagent. After receiving timely TPE, the T polyagglutination gradually disappeared. 【Conclusion】 Some bacteria, such as Streptococcus pneumoniae, may cause polyagglutination of red blood cells. The patients with suspected T polyagglutination should be diagnosed in time. For T polyagglutination patients, the minor matched plasma should be used for avoiding the random plasma with anti-T antibody transfusion.

Objective:To investigate the clinical effect of hearing aid combined with cognitive rehabilitation training in the treatment of dementia with hearing loss.Methods:Forty-six cases of dementia patients with hearing loss in Dalian Municipal Central Hospital Affiliated of Dalian Medical University from September 2019 to September 2020 were divided into two groups by random number table method. The treatment group received cognitive rehabilitation training assisted by sound amplifier, while the control group only received cognitive rehabilitation training for 7 weeks. Before and after treatment, questionnaires were used to evaluate the training effect of patients, including MMSE, HAMA, HAMD and ADL, and satisfaction questionnaire was used to evaluate the satisfaction of caregivers.Results:After treatment, the MMSE score of the treatment group was significantly higher than that of the control group: (20.22 ± 3.38) scores vs. (19.89 ± 3.10) scores ( P<0.05), and HAMA and HAMD were significantly lower than those of the control group: (11.43 ± 5.07) scores vs. (11.83 ± 4.79) scores, (8.39±3.37) scores vs. (9.67 ± 5.34) scores ( P<0.05); there was no difference in activities of daily living between the treatment group and the control group ( P>0.05); the caregiver satisfaction of the treatment group was significantly improved, compared with that of the control group ( P<0.05). Conclusions:Hearing assisted cognitive training can improve the cognitive function of patients with hearing-impaired dementia, promote mental and emotional stability, and improve the feelings of caregivers; we emphasize the importance of hearing evaluation for the elderly with dementia, and the importance of cognitive rehabilitation training with hearing assistance.

Objective:To study the application value of flexible endoscopic examination of swallowing(FEES) for the aspiration screening, the diagnosis of dysphagia and evaluation of the therapeutic effectin acute stroke patients with dysphagia.Methods:Three hundred and seventy-three patients with acute stroke who hospitalized from October 2015 to January 2020 in Dalian Municipal Central Hospital Affiliated of Dalian Medical University and underwent FEES for analyzing the characteristic performance were enrolled, and 11 cases of them were examined by video fluoroscopic swallowing study (VFSS). The results of the reliability of diagnosis dysphagia of the two methods were compared. The results of FEES for assessing the recovery effect after treatment were evaluated.Results:In 373 patients, the FEES revealed 268 cases(71.85%) of aspiration (99 cases) were recessive aspiration, which was better than that in water swallow test (50.94%, 190/373). Patients with potential cricopharyngeus achalasia got the same results through both of VFSS and FEES. FEES could provide more positive indicators and guide clinical rehabilitation treatment and objective assessment of rehabilitation effectiveness.Conclusions:Acute stroke patients with dysphagia have characteristic laryngeal performance. FEES is simple to operate and has high application value.

【Objective】 To establish a novel preparation method of cryoprecipitate coagulation factor from overcooled liquid-state plasma. 【Methods】 The fresh liquid plasma was kept at -11℃ to -13℃ for a period of time. It can remain in the liquid state with some coagulation factors generated due to supercooling. Then cryoprecipitate can be obtained from the liquid plasma by siphon method. 【Results】 The average fibrinogen content yielded in cryoprecipitate, prepared from 50 samples of 16-hour-stored fresh liquid plasma, was (186.02±22.72) mg, with the average recovery rate of (37.51±7.42) %, and the average content of coagulation FⅧ was (104.66±22.88) IU, with the average recovery rate of (46.62±5.58) %. 【Conclusion】 The cryoprecipitate coagulation factors could be obtained not only from fresh frozen-thawed plasma, but also from overcooled liquid plasma which is simple and stable, also meets the requirements of relative standards.

To study the interaction between C4b-binding protein (C4BP) and Riemerella anatipestifer (RA), we cloned duck C4BPα, conducted prokaryotic expression and prepared the polyclonal antibody by immunizing mice. Then indirect immunofluorescence assay and dot blotting hybridization assay were used to verify the interaction between C4BP and RA. The full length of duck C4BPα nucleotide sequence was 1 230 bp, with the highest similarity to chicken C4BPα (82.1%). Phylogenetic tree analysis showed that duck C4BPα and chicken C4BPα were on the same phylogenetic tree branch and the genetic evolution relationship between them was the closest. C4BPα was efficiently expressed in Escherichia coli BL21 (DE3). The recombinant proteins existed in intracellular soluble form. The titer of polyclonal antibody was more than 1:10 000 and polyclonal antibodies could specifically recognize the recombinant proteins. The results of indirect immunofluorescence assay and dot blot hybridization assay showed that RA could interact with duck C4BP. The results provide a basis to further reveal the pathogenesis of RA.
Animals ; Cloning, Molecular ; Complement C4b-Binding Protein ; chemistry ; genetics ; metabolism ; Ducks ; classification ; genetics ; microbiology ; Gene Expression Regulation ; Mice ; Phylogeny ; Riemerella ; metabolism

Objective:To study the effect of small interfering RNA (siRNA) silencing high mobility group box 1 (HMGB1) on the biological characteristics of hep-2/v resistant cells line of human laryngeal squamous cell carcinoma (HSCC).Methods:In Dalian Municipal Central Hospital, siRNA-HMGB1 was transfected into Hep-2/v cells. The expression of HMGB1 and MDR1 protein were detected by Western blot and cell immunofluorescence, cell proliferation activity was detected by CCK-8, and cell cycle was detected by flow cytometry.Results:Compared with that of the negative control group and the blank control group, the proliferation ability of the positive control group decreased by 20.18% at 24 h ( P<0.01) and 21.92% at 48 h ( P<0.05). The proportion of G 1 cells increased obviously. The expression of HMGB1 protein decreased 41.38% ( P<0.01), the expression of HMGB1 protein decreased 26.77% ( P<0.05). Conclusions:Reduction of HMGB1 expression in hep-2/v cells can inhibit cell proliferation and reduce drug resistance.